Sea cucumber active peptide, preparation method and use for reducing blood sugar

Abstract

The application discloses a sea cucumber active peptide, a preparation method and a blood glucose reduction application, belongs to the field of biological medicines, and the amino acid sequence of the active peptide is Thr-Ser-Pro-Gly-Leu-Gly-Lys-Gly-Phe. The preparation method comprises the following steps: putting chopped sea cucumbers into a reaction container, adding a compound protease for enzyme hydrolysis; enzyme inactivation and centrifugation are performed, and the supernatant is taken; the supernatant is subjected to ultrafiltration through an ultrafiltration membrane with a molecular weight cut-off of 1000 Da, the obtained filtrate is dried to obtain a sea cucumber oligopeptide powder, and Sephadex G25F gel chromatography column separation and purification and C18 reverse phase chromatography column further purification are performed. Experiments show that the active peptide can relieve STZ-induced pancreatic beta cell damage.

Description

Technical Field

[0001] This invention belongs to the field of medicine and relates to a sea cucumber active peptide, its preparation method, and its use in lowering blood sugar. Background Technology

[0002] Diabetes mellitus is a common chronic metabolic disease, and the number of patients worldwide continues to rise. Its pathogenesis is complex, mainly related to an imbalance in blood glucose regulation caused by insufficient insulin secretion or insulin resistance. Currently, there are various blood glucose-lowering treatment options, but all have certain limitations.

[0003] As the only tissue in the human body capable of secreting insulin, the treatment of pancreatic beta cells is fundamental to blood sugar control. From the perspective of overall diabetes treatment, the functional state of beta cells directly determines the course of the disease. At the time of diagnosis of type 2 diabetes, patients often have already lost more than half of their beta cell function, and traditional treatments struggle to prevent this progressive decline. If this decline can be slowed in the early stages of the disease through intensive blood sugar control or the use of cytoprotective drugs, it can not only significantly reduce the risk of long-term complications but also potentially achieve clinical remission, allowing some patients to enter a stable period where medication is no longer required. Summary of the Invention

[0004] To address the aforementioned problem of alleviating pancreatic β-cell damage, in a first aspect, according to some embodiments of this application, the active peptide has the amino acid sequence shown in SEQ ID NO.1.

[0005] In a second aspect, according to the method for preparing the active peptide in some embodiments of this application, including...

[0006] The chopped sea cucumber was placed in a reaction vessel and a complex protease was added for enzymatic hydrolysis.

[0007] Inactivate the enzyme, centrifuge, and collect the supernatant;

[0008] The supernatant was passed through an ultrafiltration membrane with a molecular weight cutoff of 1000 Da, and the resulting filtrate was dried to obtain sea cucumber oligopeptide powder.

[0009] The amount of complex protease used is 2% to 3% of the sea cucumber's weight;

[0010] Among them, the mass ratio of papain to alkaline protease in the complex protease is 1:1;

[0011] The enzymatic hydrolysis pH value is 7.0~8.0, the enzymatic hydrolysis temperature is 50~55℃, and the enzymatic hydrolysis time is 4~6h.

[0012] According to the preparation method of active peptides in some embodiments of this application, sea cucumber oligopeptides were separated and purified using a 15mm×300mm Sephadex G25F gel chromatography column. The elution solvent was deionized water, the elution flow rate was 1mL / min, the absorbance was detected at 220nm, and the elution peak with a retention time of 11-12min was collected.

[0013] Further purification was performed using a 250 mm × 4.6 mm, 5 μm C18 reversed-phase column. Mobile phase A was 0.1% trifluoroacetic acid aqueous solution, and mobile phase B was 0.1% trifluoroacetic acid acetonitrile solution. The gradient elution program was: 0–15 min 5–20% B, 15–30 min 20–30% B, 30–40 min 30–50% B, flow rate 0.8 mL / min, detection wavelength 220 nm, column temperature 35 °C. Fractions with a retention time of 30–31 min were collected.

[0014] According to the preparation method of the active peptide in some embodiments of this application, enzyme inactivation includes heating in a water bath at 90-95°C for 10-15 minutes to inactivate the enzyme.

[0015] According to the method for preparing active peptides in some embodiments of this application, centrifugation includes centrifuging at 8000 rpm for 10 min.

[0016] On a third-party level, the active peptides are prepared according to the preparation methods of the active peptides in some embodiments of this application.

[0017] In a fourth aspect, the use of the active peptides described in some embodiments of this application in the preparation of treatments for pancreatic β-cell damage diseases.

[0018] The use of the active peptides described in some embodiments of this application in the preparation of drugs for lowering blood sugar.

[0019] The use of the active peptides described in some embodiments of this application in the preparation of medicaments for the prevention, improvement or treatment of diabetes.

[0020] The use of the active peptides described in some embodiments of this application in the preparation of foods or health products that improve pancreatic β-cell damage, improve blood sugar, or improve diabetes.

[0021] Beneficial effects: Experiments show that sea cucumber oligopeptides can alleviate STZ-induced pancreatic β-cell damage in this invention. Detailed Implementation

[0022] Example 1: This disclosure describes the preparation of sea cucumber oligopeptides (SCP) using an enzymatic hydrolysis process. The method is as follows: Fresh sea cucumber raw materials are selected, washed with clean water, and chopped. The chopped sea cucumber is placed in a reaction vessel, and a complex enzyme of papain and alkaline protease (2-3% by weight of the sea cucumber) is added, wherein the mass ratio of papain to alkaline protease is 1:1. The pH of the reaction system is adjusted to 7.0-8.0, and enzymatic hydrolysis is performed in a constant temperature water bath at 50-55℃ for 4-6 hours. After enzymatic hydrolysis, the reaction vessel is placed in a water bath at 90-95℃ for 10-15 minutes to inactivate the enzyme. The hydrolysate is then centrifuged at 8000 rpm for 10 minutes, and the supernatant is collected. The supernatant is purified by ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 1000 Da. The filtrate is dried to obtain sea cucumber oligopeptide powder.

[0023] Furthermore, the above-mentioned sea cucumber oligopeptide powder was dissolved in deionized water and purified by separation using a 15mm×300mm Sephadex G25F gel chromatography column. The elution solvent was deionized water, the elution flow rate was 1mL / min, the absorbance was detected at 220nm, and the elution peak with a retention time of 11-12min was collected. Further purification was performed using a C18 reversed-phase column (250 mm × 4.6 mm, 5 μm). Mobile phase A was 0.1% trifluoroacetic acid aqueous solution, and mobile phase B was 0.1% trifluoroacetic acid acetonitrile solution. The gradient elution program was: 0-15 min 5-20% B, 15-30 min 20-30% B, 30-40 min 30-50% B, flow rate 0.8 mL / min, detection wavelength 220 nm, and column temperature 35 ℃. The fraction with a retention time of 30-31 min was collected and freeze-dried to obtain the active peptide fragment SEQ ID NO.1: Thr-Ser-Pro-Gly-Leu-Gly-Lys-Gly-Phe from sea cucumber egg oligopeptides.

[0024] Based on the above-mentioned active peptides, the preparation method of the composition with blood sugar lowering effect disclosed herein uses raw materials including 10 kg of mulberry leaves, 8 kg of Astragalus membranaceus, 6 kg of kudzu root, 5 kg of mulberry, 5 kg of Polygonatum odoratum, 3 kg of Dendrobium officinale, 5 kg of Poria cocos, 6 kg of wolfberry and 6 kg of L-arabinose.

[0025] The raw materials also include sea cucumber, and 10 kg of sea cucumber oligopeptides prepared from sea cucumber are used in the composition of this invention.

[0026] The specific preparation method includes the following steps:

[0027] S1: Raw material pretreatment: Wash, dry and crush the plant raw materials; wash and chop the sea cucumber.

[0028] S2: Extraction and preparation: Plant raw materials were extracted using a water extraction process to obtain plant extract powder PE, and sea cucumbers were prepared into sea cucumber oligopeptides SCP using the above-mentioned enzymatic hydrolysis process.

[0029] S3: Mixing and blending: Mix plant extract powder (PE), sea cucumber oligopeptide (SCP), and L-arabinose to obtain a composition.

[0030] In step S1, the plant materials, including mulberry leaves, Astragalus membranaceus, kudzu root, mulberry, Polygonatum odoratum, Dendrobium officinale, Poria cocos, and wolfberry, are washed, dried, and then pulverized into powder of appropriate particle size. In this embodiment, the mulberry leaves are dried until the moisture content is below 10%. In this embodiment, the raw materials are pulverized through a 60-mesh sieve.

[0031] In step S2, water extraction is used to extract the pretreated raw materials. The plant materials are added to 8-10 times their weight of deionized water, placed in a reaction vessel, and refluxed at 80-90°C for 2-3 times, each time for 1-2 hours.

[0032] Specifically, the steps include: for the first extraction, reflux extraction at 85°C for 1.5 hours, followed by filtration to obtain the first extract; for the second extraction, adding 8 times the mass of deionized water to the filter residue, reflux extraction at 80°C for 1 hour, followed by filtration to obtain the second extract; for the third extraction, adding 8 times the mass of deionized water to the filter residue, reflux extraction at 90°C for 1 hour, followed by filtration to obtain the third extract; combining the three extracts, filtering to remove impurities, and drying to obtain plant extract powder (PE). Filtration is preferably performed using a plate and frame filter press.

[0033] Comparative Examples E1~E8: Mulberry leaf extract, Astragalus membranaceus extract, Pueraria lobata extract, mulberry extract, Polygonatum odoratum extract, Dendrobium officinale extract, Poria cocos extract, and Lycium barbarum extract were obtained by extracting mulberry leaf extract, Astragalus membranaceus extract, Pueraria lobata extract, mulberry extract, Polygonatum odoratum extract, Dendrobium officinale extract, Poria cocos extract, and Lycium barbarum extract, respectively, according to the plant raw material extraction process in step S2 of Example 1. These extracts were named extracts E1~E8.

[0034] Experiment 1: Pancreatic β-cell intervention experiment, used to verify the effect of promoting insulin secretion and the synergistic effect of Astragalus membranaceus, mulberry and Dendrobium officinale in promoting insulin secretion.

[0035] The cell line used was the rat pancreatic islet β cell line INS-1. There were blank control group (DMEM medium), Astragalus membranaceus extract group (E2: 400 μg / mL), mulberry extract group (E4: 400 μg / mL), Dendrobium officinale extract group (E3: 400 μg / mL), and synergistic group (E2+E4+E3, 200 μg / mL each). Each group had 3 replicates.

[0036] Extracts E2, E3, and E4 were prepared according to the method in Example 1 and diluted to a concentration of 400 μg / mL using DMEM medium. INS-1 cells were seeded in 96-well plates (5 × 10⁻⁶ cells / well). 3 Cells / well were cultured at 37℃ and 5% CO2 for 24 h, then the medium was replaced with the corresponding extract, and the cells were cultured for another 48 h. Cell proliferation rate and proinsulin convertase activity were calculated, and the results are shown in Table 1.

[0037] Detection method: CCK-8 method, measuring absorbance at 450nm.

[0038] Testing indicators:

[0039] Calculate cell proliferation rate: Proliferation rate = (Experimental group absorbance - Blank group absorbance) / Blank group absorbance × 100%

[0040] Proinsulin convertase activity: Collect cell lysates and detect according to the kit instructions (unit: U / mg protein).

[0041] GLP-1 secretion: Collect culture supernatant and detect GLP-1 concentration (unit: pg / mL) using an ELISA kit.

[0042] Insulin secretion: Collect the culture supernatant and use an ELISA kit to detect insulin concentration (unit: mU / L).

[0043] Table 1

[0044]

[0045] Note: Compared with the blank control group, *P<0.05, **P<0.01; compared with each individual extract group, △P<0.05; data are mean ± standard deviation (n=3).

[0046] Table 1 shows that the Astragalus membranaceus extract group had a significantly increased cell proliferation rate (P<0.01) and a slightly increased insulin secretion (P<0.05), but no significant changes in proinsulin convertase activity and GLP-1 concentration (P>0.05). The mulberry extract group had significantly increased proinsulin convertase activity (P<0.01) and significantly increased insulin secretion (P<0.01), but no significant changes in cell proliferation rate and GLP-1 concentration (P>0.05). The Dendrobium officinale extract group had significantly increased GLP-1 concentration (P<0.01) and significantly increased insulin secretion (P<0.01), but no significant changes in cell proliferation rate and proinsulin convertase activity (P>0.05). The synergistic group had significantly higher insulin secretion than any of the individual groups (P<0.01), confirming that the three extracts significantly enhance insulin secretion through a synergistic mechanism of "proliferation-synthesis-secretion stimulation."

[0047] Experiment 2: Improving Insulin Resistance Experiment, used to verify the synergistic effect of wolfberry, Solomon's seal, and poria in improving insulin resistance.

[0048] use Adipocytes were selected from the following groups: normal control group (no insulin resistance induced, DMEM medium), model control group (palmitic acid induced insulin resistance, DMEM medium), Lycium barbarum extract group (model group + E8: 200 μg / mL), Polygonatum odoratum extract group (model group + E5: 200 μg / mL), Poria cocos extract group (model group + E7: 200 μg / mL), Polygonatum odoratum + Poria cocos synergistic group (model group + E5 + E7, 200 μg / mL each), and the three-synergistic group (model group + E8 + E5 + E7, 200 μg / mL each). Each group had 3 replicates.

[0049] The extract was prepared according to the method in Example 1 and diluted to a concentration of 200 μg / mL in DMEM medium containing 10% FBS. Mature 3T3-L1 adipocytes were cultured in medium containing 0.5 mmol / L palmitic acid for 48 h to construct an insulin resistance model (verification: glucose uptake after insulin stimulation was ≥40% lower than that in the normal group). After the model was established, the medium was replaced with the corresponding extract and the intervention was carried out for 48 h.

[0050] Western blot analysis was used to assess the phosphorylation level of IRS-1 by detecting the gray value ratio of p-IRS-1 (Tyr632) / IRS-1.

[0051] Western blot analysis was used to assess NGF pathway activity by detecting the expression levels (grayscale values) of NGF and TrkA proteins.

[0052] After insulin stimulation (100 nmol / L) for 30 min, the uptake of 2-NBDG (fluorescent glucose analogue) was measured using a kit, and the glucose uptake rate was calculated. The results are shown in Table 2.

[0053] Table 2

[0054]

[0055] Note: Compared with the blank control group, *P<0.05, **P<0.01; compared with each individual extract group, △P<0.05; data are mean ± standard deviation (n=3).

[0056] Table 2 shows that the p-IRS-1 / IRS-1 ratio in the Lycium barbarum extract group was significantly higher than that in the model group (P<0.01), and the glucose uptake rate was also significantly higher (P<0.01), but there were no significant changes in NGF and TrkA protein expression (P>0.05). The gray values ​​of NGF and TrkA proteins in the Polygonatum odoratum extract group were significantly higher than those in the model group (P<0.01), and the glucose uptake rate (62.3%±4.8%) was significantly higher (P<0.01), but the p-IRS-1 / IRS-1 ratio did not change significantly (P>0.05). The glucose uptake rate in the Poria cocos extract group was slightly higher than that in the model group (P>0.05), while there were no significant changes in IRS-1 phosphorylation and the NGF pathway (P>0.05), indicating that the effect of Poria cocos extract alone was weak. The gray values ​​of NGF and TrkA proteins in the Polygonatum odoratum + Poria cocos synergistic group were significantly higher than those in the Polygonatum odoratum alone group (P<0.05), and the glucose uptake rate (78.9%±6.1%) was significantly higher than that in the Polygonatum odoratum alone group (62.3%±4.8%, P<0.01), indicating that Poria cocos extract can enhance the activation effect of Polygonatum odoratum on the NGF pathway. The glucose uptake rate of the synergistic group (92.6%±7.3%) was close to that of the normal group (100%) and significantly higher than that of the other groups (P<0.01), indicating that the three groups synergistically improve insulin resistance by increasing insulin receptor activity, upregulating NGF pathway activity, and improving water metabolism.

[0057] Experiment 3: Inhibitory activity of mulberry leaf extract against glycosidases

[0058] Mulberry leaf extract was prepared according to the method in Example 1, and diluted to concentrations of 100, 200, and 400 μg / mL using pH 6.8 phosphate-buffered saline (PBS). A blank control group (PBS replacing the extract), a positive control group (acarbose 100 μg / mL), and various concentration groups of mulberry leaf extract were set up, with three replicates for each group. 50 μL of different concentrations of mulberry leaf extract were added to each well of a 96-well plate. Incubate with 0.1 U / mL of glucosidase solution at 37°C for 15 min; then add 50 μL of p-NPG solution (5 mmol / L) and incubate at 37°C for 30 min; finally, add 50 μL of 0.1 mol / L Na2CO3 to terminate the reaction.

[0059] The absorbance at 405 nm was measured using an ELISA reader (A); the glycosidase inhibition rate was calculated as: inhibition rate = (A value of blank group - A value of experimental group) / A value of blank group × 100%, and the half-maximal inhibitory concentration (IC50) of mulberry leaf extract was also calculated. ).

[0060] With increasing concentration of mulberry leaf extract, the inhibition rate of α-glucosidase significantly increased, reaching 78.3% ± 5.2% in the 400 μg / mL group, which showed no significant difference from the positive control group (acarbose 100 μg / mL, inhibition rate 82.5% ± 4.8%) (P>0.05). The effect of mulberry leaf extract on α-glucosidase... The concentration was 125.6 μg / mL ± 12.3 μg / mL, confirming that it has potent, dose-dependent glycosidase inhibitory activity, which can reduce the breakdown and absorption of carbohydrates in the intestine.

[0061] Experiment 4: Intervention Experiment of Pancreatic β Cells under High Glucose Environment

[0062] Pancreatic β-cells were prepared in three replicates: a normal glucose control group (5.6 mmol / L glucose, DMEM medium), a high glucose model group (25 mmol / L glucose, DMEM medium), and various concentrations of kudzu root extract (high glucose medium + E6: 100 / 200 / 400 μg / mL). Kudzu root extract was prepared according to the method in Example 1, and diluted to concentrations of 100 μg / mL, 200 μg / mL, and 400 μg / mL in DMEM medium containing 25 mmol / L high glucose. INS-1 cells were seeded in 6-well plates (2 × 10⁶ cells / well). 5 Cells / well), cultured for 24 h, then the corresponding medium was replaced, and the intervention lasted for 72 h. Annexin V-FITC / PI double staining was performed, and the percentage of apoptotic cells was detected by flow cytometry.

[0063] Collect cell lysates and test Caspase-3 activity (unit: U / mg protein) according to the kit.

[0064] Pancreatic β-cell function: Collect culture supernatant and detect insulin secretion (mU / L).

[0065] Table 3

[0066]

[0067] Table 3 shows that the apoptosis rate of cells in the high glucose model group was significantly higher than that in the normal group (P<0.01); the apoptosis rate in the 400 μg / mL kudzu extract group was significantly lower than that in the model group (P<0.01), and the Caspase-3 activity was significantly lower in the 400 μg / mL kudzu extract group than in the model group (P<0.01), indicating that kudzu extract can significantly reduce the apoptosis rate of pancreatic β cells under high glucose conditions. Furthermore, the insulin secretion level in the high glucose model group was significantly lower than that in the normal group, while the insulin secretion level in the 400 μg / mL kudzu extract group was significantly higher than that in the model group (P<0.01) and close to the control group level, indicating that kudzu extract can protect the secretory function of pancreatic β cells under high glucose conditions.

[0068] Experiment 5: Streptozotocin (STZ)-induced mouse pancreatic β-cell damage model

[0069] Mouse pancreatic β-cells were prepared using a blank control group (high-glucose DMEM medium), a model group (high-glucose DMEM medium containing 0.5 mmol / L STZ), and various concentration groups of sea cucumber oligopeptides (high-glucose DMEM medium containing 0.5 mmol / L STZ, 100 / 200 / 400 μg / mL sea cucumber oligopeptides). Sea cucumber oligopeptides were prepared according to the method in Example 1, and high-glucose DMEM medium was used to prepare concentrations of 100 μg / mL, 200 μg / mL, and 400 μg / mL. Cells were seeded in 24-well plates at a seeding density of 5 × 10⁶ cells / well. 4 Cells were cultured in basal medium at 37°C for 24 hours until 70%-80% cell adhesion and confluence. Except for the control group, all other groups were incubated with STZ (final concentration 0.5 mmol / L) for 24 hours to induce pancreatic β-cell damage. Subsequently, each intervention group was given the corresponding concentration of sea cucumber oligopeptide, while the control and model groups were given an equal volume of basal medium and cultured for another 72 hours. Cell viability was assessed using the CCK-8 assay, insulin secretion was measured using ELISA, and the percentage of apoptotic cells was determined by flow cytometry.

[0070] Table 4

[0071]

[0072] As shown in Table 4, the sea cucumber oligopeptide Thr-Ser-Pro-Gly-Leu-Gly-Lys-Gly-Phe peptide can alleviate STZ-induced pancreatic β-cell damage.

[0073] Experiment 6: Diabetic Rats Experiment

[0074] Animal model used: SPF-grade male SD rats (200-220g) were used to induce a type 1 diabetes model (blood glucose ≥16.7mmol / L for successful modeling) with streptozotocin (STZ, 60mg / kg intraperitoneal injection), totaling 60 rats. Complete formulation (containing all four functional groups): Plant extract powders (E1-E8), sea cucumber oligopeptides (SCP), and L-arabinose were mixed according to the proportions in Example 1 and prepared into a 1g / mL suspension with physiological saline (administered via gavage at 10mL / kg of rat body weight). Removal of secretion-promoting groups: The complete formulation removed Astragalus membranaceus, mulberry, and Dendrobium officinale extracts. Removal of sensitizing groups: The complete formulation removed Lycium barbarum, Polygonatum odoratum, and Poria cocos extracts. Removal of absorption-inhibiting groups: The complete formulation removed mulberry leaf extract and L-arabinose. Removal of protective groups: The complete formulation removed sea cucumber oligopeptides and Pueraria lobata extract. Group design: A normal control group (no modeling, administered saline by gavage), a model control group (modeling, administered saline by gavage), a complete formulation group, a group lacking secretion promotion, a group lacking sensitization, a group lacking absorption inhibition, and a group lacking protection were established, with 10 rats in each group. Rats were administered saline by gavage for 8 consecutive weeks. Blood glucose-related indicators: Fasting blood glucose (FBG) was measured weekly, and 2-hour postprandial blood glucose (2hPBG) and HbA1c were measured at week 8. Insulin-related indicators: Serum insulin (INS) and insulin resistance index (HOMA-IR = FBG × INS / 22.5) were measured at week 8. Pancreatic islet function: Rats were sacrificed at week 8, and pancreatic tissue was collected. Islet morphology was observed by HE staining, and the number of pancreatic β cells was calculated. Safety indicators: Serum ALT and AST were measured at week 8. Results are shown in Table 5.

[0075] Table 5

[0076]

[0077] Table 5 shows that fasting blood glucose, 2hPBG, and HbA1c in the model control group were significantly higher than those in the normal control group (P<0.01), indicating that the diabetes model was successfully established and blood glucose control was extremely poor. After 8 weeks of intervention, the three indicators in the complete formula group were significantly lower than those in the model control group (P<0.01) and close to the levels of the normal control group, demonstrating a strong and stable hypoglycemic effect. The intervention effects of each functional group were significantly weaker than those in the complete formula group (P<0.05). Among them, the group lacking absorption inhibition showed the smallest decrease in 2hPBG, and the group lacking protection showed the smallest decreases in FBG and HbA1c, indicating that each functional group played an irreplaceable role in different dimensions of blood glucose control (postprandial blood glucose and long-term blood glucose).

[0078] Meanwhile, in the model control group, INS decreased to 8.2±1.5 mU / L, and HOMA-IR increased to 8.6±0.7, indicating severe impairment of pancreatic β-cell function, insufficient insulin secretion, and severe insulin resistance (P<0.01). After intervention, both indicators in the complete formula group were significantly improved compared to the model control group (P<0.01), indicating that the complete formula can both promote insulin secretion and effectively improve insulin resistance. The deficient groups showed varying degrees of abnormal indicators: the insulin secretion-promoting group had an INS level of 14.2±2.1 mU / L, significantly lower than the complete formula group (P<0.05), confirming that the insulin secretion-promoting group was crucial for increasing INS levels; the insulin sensitizing group had a HOMA-IR level of 5.8±0.5, significantly higher than the complete formula group (P<0.05), reflecting the core role of the sensitizing group in improving insulin resistance; the insulin protection group had an INS level of 11.3±1.6 mU / L and a HOMA-IR level of 6.8±0.7, both the worst, indicating that protecting islet cells can simultaneously maintain insulin secretion capacity and improve insulin sensitivity; the insulin absorption-inhibiting group had INS and HOMA-IR levels of 13.8±2.0 mU / L and 6.2±0.6, respectively, which were better than the model control group, but still significantly worse than the complete formula group (P<0.05). Furthermore, in the normal control group, the islets of Langerhans were morphologically intact, structurally clear, with 132±15 β-cells per islet, tightly packed, and functionally normal. In the model control group, the islets of Langerhans in rats showed significant atrophy and structural disorder, with only 45±8 β-cells per islet, a decrease of 65.9% compared to the normal control group (P<0.01). Extensive apoptosis of β-cells and severe functional decline were observed. In the complete formula group, the islet morphology of rats was largely restored, the degree of atrophy was significantly reduced, and the number of β-cells increased to 108±12 per islet, an increase of 140% compared to the model control group (P<0.01). Furthermore, the β-cells were more neatly arranged, confirming that the complete formula has a significant protective and repairing effect on pancreatic β-cells. The number of pancreatic β cells in each of the functional deficient groups was significantly less than that in the intact group (P<0.05): 72±10 cells / island in the secretion-promoting group, 68±9 cells / island in the sensitizing group, 70±11 cells / island in the absorption-inhibiting group, and only 58±8 cells / island in the protection group. The number of pancreatic β cells in the protection group was closest to that in the model control group, further verifying the key role of the pancreatic islet cell protection group (sea cucumber oligopeptide, kudzu root extract) in maintaining β cell number and repairing islet structure.

[0079] In the model control group, ALT, AST, Scr, and BUN levels were significantly higher than those in the normal control group (P<0.01), indicating that liver and kidney function impairment had occurred in the diabetic model rats. The complete formula group showed significant protective effects on the liver and kidneys after 8 weeks of intervention, with no obvious toxic side effects. Among the functional deficiencies, the protective group had the worst liver and kidney function indicators, with little difference from the model control group (P>0.05), indicating that the pancreatic islet cell protection group not only protected the islets but also had a synergistic protective effect on the liver and kidneys. While the liver and kidney function indicators in other functional deficiencies groups (deficiency of secretion-promoting group, deficiency of sensitizing group, and deficiency of absorption-inhibiting group) were better than those in the model control group, they were still slightly higher than those in the complete formula group (P>0.05), further demonstrating the safety advantage of the complete formula.

[0080] In summary, the results of the diabetic rat experiments show that the complete formula can improve diabetic symptoms from four dimensions—lowering blood glucose levels, improving insulin function, repairing pancreatic islet structure, and protecting liver and kidney function—through the synergistic effect of promoting insulin secretion, improving insulin sensitivity, inhibiting intestinal carbohydrate absorption, and protecting pancreatic islet cells. Moreover, the effect is significantly better than the formula that lacks any one of the functional groups.

[0081] This invention discloses a hypoglycemic formula and its application. The formula comprises water, mulberry leaves, sea cucumber oligopeptides, Astragalus membranaceus, kudzu root, mulberry, Polygonatum odoratum, Dendrobium officinale, Poria cocos, wolfberry, and L-arabinose. The components achieve hypoglycemic effects through multi-target synergistic effects. For example, alkaloids in mulberry leaves inhibit glycosidase activity, sea cucumber oligopeptides lower blood sugar and protect the liver and kidneys, various plant components promote insulin secretion and improve insulin sensitivity, and L-arabinose inhibits sucrose absorption. Compared with existing hypoglycemic methods, this invention's formula is innovative in its multi-target synergistic effect and natural safety. This invention also includes a preparation method based on this formula, comprising steps such as raw material pretreatment, extraction, sea cucumber oligopeptide preparation, mixing and blending, sterilization and filling, etc. The process is reasonable and can be mass-produced.

[0082] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An active peptide, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

1.

2. A method for preparing an active peptide, characterized in that, include The chopped sea cucumber was placed in a reaction vessel and a complex protease was added for enzymatic hydrolysis. Inactivate the enzyme, centrifuge, and collect the supernatant; The supernatant was passed through an ultrafiltration membrane with a molecular weight cutoff of 1000 Da, and the resulting filtrate was dried to obtain sea cucumber oligopeptide powder. The amount of complex protease used is 2% to 3% of the sea cucumber's weight; Among them, the mass ratio of papain to alkaline protease in the complex protease is 1:1; The enzymatic hydrolysis pH value is 7.0~8.0, the enzymatic hydrolysis temperature is 50~55℃, and the enzymatic hydrolysis time is 4~6h.

3. The method according to claim 2, characterized in that, Sea cucumber oligopeptides were separated and purified using a 15 mm × 300 mm Sephadex G25F gel chromatography column. The elution solvent was deionized water, the elution flow rate was 1 mL / min, the absorbance was detected at 220 nm, and the elution peak with a retention time of 11-12 min was collected. Further purification was performed using a 250 mm × 4.6 mm, 5 μm C18 reversed-phase column. Mobile phase A was 0.1% trifluoroacetic acid aqueous solution, and mobile phase B was 0.1% trifluoroacetic acid acetonitrile solution. The gradient elution program was: 0–15 min 5–20% B, 15–30 min 20–30% B, 30–40 min 30–50% B, flow rate 0.8 mL / min, detection wavelength 220 nm, and column temperature 35 °C. Fractions with a retention time of 30–31 min were collected.

4. The method according to claim 2 or 3, characterized in that, in, Enzyme inactivation involves heating in a water bath at 90-95°C for 10-15 minutes.

5. The method according to claim 2 or 3, characterized in that, in, Centrifugation includes centrifuging at 8000 rpm for 10 minutes.

6. The active peptide prepared by any one of claims 2-5.

7. The use of the active peptide according to claim 1 or 6 in the preparation of a treatment for pancreatic β-cell damage diseases.

8. The use of the active peptide according to claim 1 or 6 in the preparation of a drug for lowering blood sugar.

9. The use of the active peptide of claim 1 or 6 in the preparation of a medicament for the prevention, improvement or treatment of diabetes.

10. The use of the active peptide of claim 1 or 6 in the preparation of food or health products that improve pancreatic β-cell damage, improve blood glucose, or improve diabetes.

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